PROJECT SUMMARY Pain (Dolor) is one of the four cardinal signs of inflammation, and often accompanies bacterial infections. Nociceptor neurons are the peripheral sensory neurons that mediate pain, which densely innervate barrier tissues including the skin and soft tissues that are exposed to pathogens. Streptococcus pyogenes is a leading cause of necrotizing fasciitis, an invasive and life-threatening form of infection, in which pain occurs early and ?out of proportion? with other symptoms. We hypothesize that pain plays a major causative role in the pathogenesis of S. pyogenes, by inducing neural mediated suppression of innate immune cell recruitment and killing of bacteria. Here, we will determine: 1) The molecular mechanisms of pain during S. pyogenes infection, with a focus on streptococcal pore-forming toxins streptolysin S (SLS) and streptolysin O (SLO), and 2) The role of nociceptors and pain signaling in regulating neutrophil function and host defense against S. pyogenes. We test the hypothesis that targeting pain signaling would lead to enhanced innate immune responses. Given the importance of pain in the diagnosis of necrotizing fasciitis, and the widespread use of analgesics, our findings connecting pain to S. pyogenes host defense could have important clinical implications. The two aims of this study leverage the complementary skills of Dr. Chiu and Dr. Wessels, combining neurobiological, immunological, and microbiological approaches to investigate the role of pain in host defense. In Specific Aim 1, we will determine the critical molecular mechanisms of pain production during S. pyogenes infection by two clinical isolates. We use isogenic mutant bacterial strains and plasmid complementation strategies together with neurobehavioral assays to determine whether SLS and SLO mediate S. pyogenes-induced pain. We will determine whether the inflammasome and IL-1? are involved in neuronal recognition of pore-forming toxins or pain signaling. In addition, we determine the effectiveness of non-steroidal anti-inflammatory drugs (NSAIDs) in impacting pain perception during S. pyogenes infection. In Specific Aim 2, we will determine how targeted ablation of TRPV1+ nociceptors using RTX treatment or Trpv1-cre/DTA mice enhances host defenses against S. pyogenes. We will determine how temporally and spatially controlled modulation of TRPV1+ neural activity using optogenetic or DREADD strategies affects the outcome of S. pyogenes infection. We will also utilize opioids to block central pain perception or Botulinum toxin to block peripheral pain signaling to determine which neural component modulates immune responses against S. pyogenes. In our analysis, we elucidate the role of nociceptor-derived neuropeptides such as CGRP in suppressing neutrophil phagocytosis and killing of S. pyogenes. This study analyzes a significant link between induction of pain signaling, neural blockade of innate immunity, and the potentiation of S. pyogenes bacterial pathogenesis. Targeting pain and neuro-immune suppression could lead to novel approaches to treatment of this and other invasive bacterial infections.